Difference: StewartShuman (3 vs. 4)

My Links

• NysbcIntro - your introduction to NYSBC
• NetLiterate - short introduction to the style of this intranet

Personal Preferences

Uncomment preferences variables to activate them (remove the #-sign). Help and details on preferences variables are available in TWikiPreferences.

• Show tool-tip topic info on mouse-over of WikiWord links, on or off:
  • #Set LINKTOOLTIPINFO = off
• Horizontal size of text edit box:
  • #Set EDITBOXWIDTH = 70
• Vertical size of text edit box:
  • #Set EDITBOXHEIGHT = 22
• Style of text edit box. width: 99% for full window width (default), width: auto to disable.
  • #Set EDITBOXSTYLE = width: 99%
• Write protect your home page: (set it to your WikiName)
  • #Set ALLOWTOPICCHANGE = StewartShuman

Related Topics

• ChangePassword for changing your password
• ChangeEmailAddress for changing your email address
• TWikiUsers has a list of other Intranet users
• UserDocumentationCategory is a list of Intranet user documentation
• UserToolsCategory lists all Intranet user tools

Grant Number: 5R01GM042498-19 Project Title: Viral RNA Modifying Enzymes PI Information: Name Email Title SHUMAN, STEWART H. s-shuman@ski.mskcc.org PROFESSOR

Abstract: DESCRIPTION (provided by applicant): Project Summary: Our long-term goals are to understand the catalytic mechanism and biological specificity of virus-encoded enzymes that modify RNA ends. We are studying viral mRNA capping and viral tRNA repair as paradigms of RNA transactions that enable virus replication. The studies of capping provide fresh insights to the evolution of a uniquely eukaryotic mRNA processing event and are opening up new approaches to antiviral drug discovery targeted at viral mRNA cap formation. The current research plan is focused on the RNA triphosphatase and RNA (guanine-N7) methyltransferase components of the poxvirus mRNA capping apparatus and includes specific aims to: (a) interrogate whether the viral capping activities are essential for poxvirus replication in cell culture; (b) map the active site of the cap methyltransferase and determine its structure with substrates and inhibitors bound; (c) evaluate a sinefungin-based transition state analog as a specific inhibitor of poxvirus cap methylation; (d) determine the structure of the viral RNA triphosphatase and identify novel inhibitors via high-throughput screening. Our studies of tRNA repair are illuminating the evolutionary transitions from RNA-world to DNA-world enzymology. This proposal focuses on bacteriophage T4 Pnkp, a bifunctional 5' kinase/3' phosphatase that heals broken tRNA ends. Pnkp functions in vivo to antagonize a host antiviral response that blocks viral protein synthesis through tRNA depletion. Our aim is to elucidate the mechanism of the 3'-phosphatase domain by capturing structures of the enzyme at different steps along the reaction pathway. Relevance: Exploitation of new molecular targets for treatment of poxvirus infections is a pressing issue, given the concerns that smallpox could be used as a bioterror weapon and the risk of complications of vaccinia infections if a prophylactic vaccination program is resumed. The outbreak of human monkeypox infections in the US in 2003 highlighted the risks of re-emergence of human poxvirus disease. mRNA capping enzyme is an attractive therapeutic target for smallpox because the active site and catalytic mechanism of the poxvirus RNA triphosphatase are completely different from that of the human RNA triphosphatase. Poxvirus cap methyltransferase is also distinguished from the human counterpart by its reliance on a unique virus-encoded regulatory subunit.

Public Health Relevance: This Public Health Relevance is not available.

Thesaurus Terms: acid anhydride hydrolase, enzyme mechanism, ligase, nucleotidyltransferase, phosphotransferase, posttranscriptional RNA processing, virus RNA, virus protein active site, messenger RNA, protein structure, virus genetics Baculoviridae, DNA virus, X ray crystallography, site directed mutagenesis, vaccinia virus

Institution: SLOAN-KETTERING INSTITUTE FOR CANCER RES 1275 YORK AVE NEW YORK, NY 10065 Fiscal Year: 2008 Department: Project Start: 01-JUL-1989 Project End: 31-MAR-2011 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: VIRA

Abstract: DESCRIPTION (provided by applicant): The 5' m7GppN cap is a distinctive feature of eukaryotic mRNA that is required for mRNA stability and translation. The mRNA cap is formed by three enzymes: RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-N7) methyltransferase. Our long-term goals are to understand the mechanisms and specificities of the capping enzymes, illuminate how capping is coupled to transcription through interactions of the capping enzymes with components of the Pol II elongation complex, and explore the capping enzymes as anti-infective drug targets. Our studies have revealed differences in the structures, mechanisms, and inhibition profiles of capping enzymes from different taxa that recommend RNA triphosphatase and cap (guanine-N7) methyltransferase as targets for antifungal/antiprotozoal drug discovery - a major "unmet need" in public health and infection control worldwide. Here we propose to uncover the basis for the exquisite sensitivity of the fungal cap guanine-N7 methyltransferase to the natural product sinefungin and to develop a bisubstrate transition-state analog built on a sinefungin scaffold. We've shown that the capping enzymes are directed to nascent mRNAs by binding to the phosphorylated carboxyl-terminal domain (CTD) of the largest subunit of RNA Pol II, which is composed of a tandem array of YSPTSPS heptapeptide repeats. Capping enzymes also interact physically with elongation factor Spt5 and the CTD kinase Cdk9. This interaction network suggests an "elongation checkpoint" that recruits the capping enzymes in a timely fashion and thereby avoid wasteful rounds of transcription of uncapped pre-mRNAs. We propose to dissect these interactions genetically and biochemically in order to test key predictions of the checkpoint model. A related goal is to explore the how the effector functions of the Pol II CTD are modulated by CTD-specific phosphatases. We focus on the essential phosphatases SpFcpl^? and Ssu72, which display a preference for Ser2-P and Ser5-P, respectively. A partial deficiency of human Fcp1 is associated with a genetic developmental disorder. We are poised to determine the atomic structure of ScFcpl^?. We have initiated a new line of research into the formation of the 2,2,7-trimethylguanosine (TMG) cap structure found on many small nuclear RNAs. We characterized Tgs1 and Tgs2 as cap-specific guanine-N2 methyltransferases. We aim to elucidate their mechanism and structure.

Public Health Relevance: This Public Health Relevance is not available.

Thesaurus Terms: acid anhydride hydrolase, enzyme mechanism, enzyme structure, messenger RNA, methyltransferase, nucleotidyltransferase, posttranscriptional RNA processing DNA directed RNA polymerase, active site, enzyme complex, fungal genetics, fungal protein, gene expression, nucleic acid structure, precursor mRNA, protein protein interaction, transcription factor Saccharomyces cerevisiae, X ray crystallography, clinical research

Institution: SLOAN-KETTERING INSTITUTE FOR CANCER RES 1275 YORK AVE NEW YORK, NY 10065 Fiscal Year: 2008 Department: Project Start: 01-MAY-1995 Project End: 30-APR-2011 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: MGB

Abstract: DESCRIPTION (provided by applicant): Breaks in the phosphodiester backbone of DNA and essential RNA molecules can lead to cell death if not repaired. This project aims to illuminate the mechanisms and structures of the DNA ligase and RNA ligase enzymes that rectify such breaks. Polynucleotide ligases catalyze the joining of a 5'-PO4 strand to a 3'-OH end via 3 chemical steps: (i) ligase reacts with ATP or NAD+ to form a covalent ligase-adenylate intermediate and release pyrophosphate or NMN; (ii) AMP is transferred from the ligase to the 5'-PO4 DNA or RNA strand to form a DNA/RNA-adenylate intermediate (AppDNA^? or AppRNA^?); (iii) ligase catalyzes attack by the 3'-OH of the nick on AppDNA^? or AppRNA^? to form a phosphodiester and release AMP. Our long-range goals are to understand how ligase reaction chemistry is catalyzed and how ligases recognize "damaged" DNA or RNA ends. We are approaching these problems using a eukaryotic virus-encoded DNA ligase (Chlorella virus DNA ligase), a bacterial NAD+-dependent DNA ligase (E. coli LigA^?), and a bacteriophage ATP-dependent RNA ligase (T4 Rnl2) as models. Rnl2 was discovered by this laboratory during the previous funding period and quickly developed into a model-of-choice for RNA repair enzymology. Rnl2 exemplifies a new and growing family of RNA ligases found in all phylogenetic domains. Our studies, which integrate biochemistry, molecular genetics, and structural biology, have revealed mechanistic principles shared by all DNA and RNA ligases, as well as the unique structural features and substrate specificities that distinguish the various branches of the polynucleotide ligase superfamily. In particular, our work indicates that progression through the sequential steps of the ligation pathway is coupled to large protein domain movements and serial remodeling of the active site. Specific aims of this proposal are: (1) To identify the functional groups of Chlorella virus DNA ligase, E. coli LigA^?, and T4 Rnl2 that contribute to DNA/RNA recognition and nucleotidyl transfer; (2) To biochemically define the interface between ligase-adenylate and nicked DNA/RNA substrates; and (3) To determine atomic structures of ligase-adenylate bound at a DNA/RNA nick and of ligases bound to the 5'-adenylated polynucleotide intermediate.

Public Health Relevance: This Public Health Relevance is not available.

Thesaurus Terms: DNA repair, enzyme mechanism, enzyme structure, phosphoester ligase, protein structure function DNA damage, adenosine triphosphate, catalyst, enzyme activity, enzyme substrate, structural biology site directed mutagenesis

Institution: SLOAN-KETTERING INSTITUTE FOR CANCER RES 1275 YORK AVE NEW YORK, NY 10065 Fiscal Year: 2008 Department: Project Start: 01-AUG-2001 Project End: 31-JUL-2009 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: MSFC

Abstract: DESCRIPTION (provided by applicant): Our long-term goals are to understand the mechanism and biological functions of DNA topoisomerase IB (ToplB^?). The ToplB^? family includes eukaryotic nuclear and mitochondrial ToplB^?, poxvirus and mimivirus topoisomerases, and the poxvirus-like topoisomerases of bacteria. ToplB^? enzymes relax DNA supercoils by breaking and rejoining one strand of the DNA duplex. They act via a transesterification mechanism involving a covalent DNA-(3'-phospho-tyrosyl)-enzyme intermediate. This laboratory uses vaccinia virus as a model system to study ToplB^?. The vaccinia-encoded ToplB^? is packaged within the virus particle, where it plays a critical role in replicative fitness by aiding viral mRNA synthesis. A distinctive feature of the poxvirus ToplB^? is its specificity in forming a covalent intermediate at a target sequence 5'-(C/T)CCTT. All poxvirus topos recognize this site, as does the homologous mimivirus ToplB^? enzyme. We hypothesize that DNA target recognition triggers the recruitment of catalytic amino acid side chains to form the ToplB^? active site. An aim of this project is to elucidate at single-atom resolution the structural basis for DNA transesterification and target site specificity and to define the conformational steps for active site assembly and supercoil relaxation. This will be accomplished by an innovative multidisciplinary approach involving DNA chemistry, protein modification with non-natural amino acids, and single-molecule studies, along with "classical" structure-guided mutagenesis and biochemistry. We also aim to dissect genetically which properties of vaccinia ToplB^? are important in vivo, by gauging the effects of biochemically characterized ToplB^? mutations on vaccinia virus replication. Relevance: Understanding the catalytic mechanism of ToplB^? is a high priority because: ToplB^? is implicated in virtually every DNA transaction in human cells; nuclear ToplB^? is the target of anticancer drugs that exert their cytotoxicity by perverting the cleavage-religation equilibrium; and ToplB^? enzymes are distributed widely in bacterial and viral pathogens, where they present untapped targets for mechanism-based anti-infective drug discovery. Exploitation of new molecular targets for treatment of poxvirus infections is a pressing issue, given the concerns that smallpox could be used as a bioterror weapon and the risk of complications of vaccinia infections if a prophylactic vaccination program is resumed.

Public Health Relevance: This Public Health Relevance is not available.

Thesaurus Terms: DNA topoisomerase, enzyme activity, enzyme mechanism, enzyme structure, vaccinia virus Mycobacterium avium, Mycobacterium smegmatis, Pseudomonas aeruginosa, active site, benzopyrene, catalyst, chemical cleavage, endodeoxyribonuclease, enzyme substrate, esterification, genetic recombination, virus replication X ray crystallography, electron microscopy

Institution: SLOAN-KETTERING INSTITUTE FOR CANCER RES 1275 YORK AVE NEW YORK, NY 10065 Fiscal Year: 2008 Department: Project Start: 01-JUL-1991 Project End: 30-JUN-2012 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: VIRA